Methods for storing conifer somatic embryo germinants

ABSTRACT

In one aspect, the present invention provides methods of storing conifer somatic embryo germinants germinated oil a sterile germination medium for delayed transplanting into a growth medium. The methods of the invention comprise the steps of: (a) placing the germinants while still on sterile germination medium into a cold environment in which the temperature is in the range of 0.5° C. to 10° C. for a time period up to six months, said germinants comprising a visible, well-defined epicotyl and radical; and (b) placing the germinants in water for a time greater than about 1 hour at a temperature below about 24° C. prior to transplantation into growth medium.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional of pending U.S. patentapplication Ser. No. 11/566,123, filed Dec. 1, 2006, which claims thebenefit of U.S. Provisional Application No. 60/753,453, filed Dec. 22,2005.

FIELD OF THE INVENTION

The present invention relates to methods for storing conifer somaticembryo germinants.

BACKGROUND OF THE INVENTION

The demand for coniferous trees, such as pines and firs, to make woodproducts continues to increase. One proposed solution to meeting thishigh demand is to identify individual trees that possess desirablecharacteristics, such as a rapid rate of growth, and produce numerous,genetically identical, clones of the superior trees by somatic cloning.

Somatic cloning is the process of producing plant embryos, in vitro,from plant cells that are not zygotes. These clones can be cultivated toyield stands, or whole forests, of conifer trees that possess thedesirable characteristic(s). One method for somatically cloning treesutilizes in vitro treatment of isolated, living, conifer tissue underconditions that promote formation of conifer somatic embryos, and thenwhole plants from the treated tissue. An explant such as an immatureseed or the embryo from an immature seed is placed on a gelledinitiation medium. This medium will usually contain plant growthhormones from the groups known as auxins and cytokinins. If initiationis successful a gelatinous mass containing multiple immature embryoswill be generated in several weeks. This mass is then removed andsubcultured on a maintenance and multiplication medium which may begelled, liquid, or some combination of these. Embryos from maintenancemedium may then be placed on a development medium that normally lacksthe auxins and cytokinins but may instead include the hormone abscisicacid. The somatic embryos develop into cotyledonary embryos with a sizeand morphology that closely resembles their mature zygotic counterparts.The embryos are then placed on a germination medium on which radical andepicotyl elongation occur over a period of several weeks. The germinantsare then removed and placed in soil for further development intoplantlets. After a period of greenhouse growth they may be outplanted.Alternatively, the embryos removed from the development medium may beplaced into manufactured seeds; e.g., as shown in Carlson et al., U.S.Pat. No. 5,236,469.

A continuing problem, however, has been that conversion percentage fromsomatic embryos to plants growing in soil has frequently been lower thandesired. In addition, a logistical problem exists for handling largenumbers of conifer somatic embryos that are not packaged intomanufactured seeds. These embryos are individually removed from thesterile development medium and placed on a germination medium. After anappropriate time the germinants are placed in a potting soil mixture forfurther growth. Typically many hundreds of thousands of germinants arepotted at once in clonal field tests. The germinants are very tender andsusceptible to damage by disease organisms such as fungi. In addition,it is preferable to outplant germinants that have similar dimensions inclonal field tests in order to obtain reliable results regarding thecharacteristics of various clones. However, a logistical challengeexists for producing a population of germinants having similardimensions at the time of outplanting.

There is therefore a continuing need for a method for storing conifergerminants that reduces the risk of contamination with microbes, isamenable to large scale production, and results in a high conversionrate after transplantation.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods of storing conifersomatic embryo germinants germinated on a sterile germination medium fordelayed transplanting into a growth medium. The methods of the inventioncomprise the steps of: (a) placing the germinants while still on sterilegermination medium into a cold environment in which the temperature isin the range of 0.5° C. to 10° C. for a time period up to 6 months, saidgerminants comprising a visible, well-defined epicotyl and radical; and(b) placing the germinants in water for a time greater than about 1 hourat a temperature below about 24° C. prior to transplantation into growthmedium.

In another aspect, the present invention provides methods of producingand storing germinants prior to transplantation into growth medium. Themethods of this aspect of the invention comprise the steps of: (a)culturing conifer somatic embryos on sterile germination medium for asufficient period of time to produce germinants, said germinantscomprising a visible, well-defined epicotyl and radical; (b) placing thegerminants while still on sterile germination medium into a coldenvironment in which the temperature is in the range of 0.5° C. to 10°C. for a time period up to 6 months, and (c) placing the germinants inwater at a temperature below about 24° C. for a time greater than about1 hour prior to transplanting the stored germinants into growth medium.

In another aspect, the present invention provides methods of increasingthe conversion percentage from somatic embryos to plants growing ingrowth medium. In accordance with this aspect of the invention themethods comprise the steps of: (a) culturing conifer somatic embryos onsterile germination medium for a sufficient period of time to producegerminants, said germinants comprising a visible, well-defined epicotyland radical; (b) placing the germinants while still on sterilegermination medium into a cold environment in which the temperature isin the range of 0.5° C. to 10° C. for a time period up to 6 months; (c)placing the germinants in water for a time greater than about 1 hour ata temperature below about 24° C.; and (d) removing the germinants fromthe water and transplanting them into growth medium for further growth.

In yet another aspect, the present invention provides methods ofproducing a synchronized population of somatic embryo germinants inpreparation for transplantation. In accordance with this aspect of theinvention the methods comprise the steps of, (a) culturing somaticembryos on sterile germination medium for a sufficient period of time toproduce germinants having a desired dimension; (b) selecting a pluralityof germinants having the desired dimension; (c) storing the selectedgerminants while still on sterile germination medium in a coldenvironment in which the temperature is in the range of 0.5° C. to 10°C. for a time period up to 6 months; and (d) placing the storedgerminants in water for a time greater than about 1 hour at atemperature below about 24° C. prior to planting the selected germinantsinto growth medium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

As used herein, the term “cotyledonary embryo” means an embryo thatpossesses one or more cotyledons.

As used herein, the term “somatic embryo” refers to a plant embryo thatdeveloped in vitro from a plant cell, or tissue, that is not a zygote.

As used herein, the term “embryogenic tissue” refers to any tissue,derived from a conifer which is capable of producing one or more conifercotyledonary somatic embryos, including, for example, conifer embryonalsuspensor masses.

As used herein the term “germinant” refers to an immature plant thatpossesses a well developed radical and cotyledonary structure with agrowing epicotyl, both readily apparent t) the naked eye, and ready forplanting in soil. For example, the germinants typically have an epicotylof about 10 mm or greater.

As used herein, the term “converted embryo” is an embryo that hasgerminated and been established as a plant growing in soil.

As used herein, the term “synchronized population of germinants” refersto a stock of germinants having a target specification at outplanting.The germinants may be cultured to a desired target specification, suchas having a desired dimension, (e.g., an epicotyl stem length of atleast 10 mm).

Unless stated otherwise, all concentration values that are expressed aspercentages are weight per volume percentages.

The present invention provides methods for storing conifer somaticembryo germinants. The methods each include the step of placing thegerminants while still on germination medium into a cold environment fora time period of up to 6 months (such as, for example, from at least 2weeks to 24 weeks, or such as from 4 weeks to 10 weeks), followed by thestep of placing the germinants in water for a time greater tan about 1hour up to from one to several weeks (such as, for example from severalhours up to one week) prior to transplantation into a growth medium. Ithas been observed that conifer germinants may be stored up to 4 weeks ormore prior to planting in growth medium when stored according to themethods of the invention without loss of post-transplant vigor.

The storage methods of the invention are particularly advantageous inthat they make it possible to produce a synchronized population ofgerminants in preparation for transplantation. The synchronizedpopulation of germinants are produced by culturing germinants to adesired target specification, and storing the germinants in a coldenvironment on sterile germination medium as they become available overa period of days or weeks. The stored germinants are then placed intowater for a time greater than about 1 hour at a temperature below about24° C. prior to planting. Consequently, the storage methods of theinvention allow for a longer embryo production window (e.g. severalweeks to several months) in which to obtain a desired population ofgerminants, which can then be outplanted within a narrow time frame(e.g. one day up to two weeks), or during a winter dormant period,thereby resulting in a synchronized population at outplanting. Theability to synchronize the production of germinants in preparation fortransplantation is particularly useful in the context of planting clonalfield tests.

The methods of the present invention can be used to store conifersomatic embryo germinants from any conifer species, such as members ofthe family Pinacea, including members of the genus Pinus (e.g., loblollypine (Pinus taeda)), or such as members of the genus Pseudotsuga (e.g.,Douglas-fir (Pseudotsuga menziesii)).

The germinants may be produced by any method that yields conifer somaticembryo germinants. For example, germinants may be produced by firstculturing conifer somatic embryos by known protocols, such as themethods described in U.S. Pat. Nos. 6,134,830 and 5,563,061. After theembryos have reached a mature cotyledonary stage they are then placed ona germination medium under environmental conditions of 21° C. to 27° C.for a sufficient period of time for an epicotyl and radical to develop.This time period may range between 4 and 14 weeks, depending on theparticular species and genotype. More typically it is between 8 and 12weeks. A suitable light/dark photoperiod may be used, such as a 24 hourphotoperiod.

The germination medium typically has no exogenous hormones, a lowerednitrogen content and a reduced level of osmoticants. The germinationmedium typically contains agar and is sterilized prior to use. Thegerminants may be germinated under suitable conditions on solidgermination media contained in any suitable container, such as in Petridishes stored in germination boxes.

An example of a suitable germination medium for Douglas-fir (Pseudotsugamenziesii) is provided below in TABLE 1.

TABLE 1 Pseudotsuga menziesii BM_(G) Germination Media ConstituentConcentration, mg/L NH₄NO₃ 206.3 KNO₃ 1170.0 CaCl₂•6H₂O 220.0 KH₂PO₄85.0 MgSO₄•7H₂O 185.0 MnSO₄•H₂O 8.45 ZnSO₄•7H₂O 4.30 CuSO₄•5H₂0 0.013FeSO₄•7H₂0 13.93 Na₂EDTA 18.63 H₃BO₃ 3.10 NaMoO₄•2H₂O 0.125CoCl_(2•)6H₂0 0.0125 KI 0.42 myo-Inositol 100.0 Thiamine•HCL 1.00Nicotinic acid 0.50 Pyridoxine•HCL 0.50 Glycine 2.00 L-Glutamine 450.0Sugar 20,000 pH 5.7 Activated charcoal 2500 Tissue culture agar 8000

In accordance with the methods of the invention, once developed, thegerminants, while still on the sterile germination medium, are placedinto a cold environment in which the temperature is in the range ofabout 0.5° C. to about 10° C., preferably from about 4° C. to about 8°C. Preferably the container comprising the germinantes on thegermination media is placed into the cold storage environment withoutany manipulation of the germinants required.

The germinants may be stored in the cold environment untiltransplantation into growth medium for at least two weeks up to about 6months (such as from 1 week to 20 weeks, or more preferably from about 2weeks to about 4 weeks) without loss of post-transplant vigor, asdescribed in EXAMPLES 1 and 2.

After the desired time of storage on germination medium and prior totransplant, the germinants are removed from the germination medium andplaced in deionized water in sealed dishes. In some embodiments of themethod, the germinants are transferred under sterile conditions, such asunder a laminar flow hood. In other embodiments of the method, thegerminants are transferred under non-sterile conditions. The germinantsare stored in water for a time greater than about 1 hour at atemperature below about 24° C. Preferred temperatures are below 10° C.,such as from about 0.5° C. to about 5° C. Improved rates of posttransplant survival have been found for germinants stored in water for aperiod greater than 1 hour to about three weeks, as described in EXAMPLE2.

The germinants stored in accordance with the method of the invention areeventually transferred to growth medium for further growth whicheventually may result in conifer trees. The growth medium may be anysuitable medium, such as potting soil, or a mixture of peat, vermiculateand perlite. The potted germinants are typically grown in a greenhouseand maintained at 24° C. for a 16 hour light period and at 18° C. for an8 hour dark period. The plants may be compared and rated for survivalpercentage as described in EXAMPLES 1 and 2.

In another aspect, the present invention provides methods of increasingthe conversion percentage from somatic embryos to plants growing ingrowth medium. The method according to this aspect of the inventioncomprise the steps of: (a) culturing conifer somatic embryos on sterilegermination medium for a sufficient period of time to producegerminants, said germinants comprising a visible, well-defined epicotyland radical; (b) placing the germinants while still on sterilegermination medium into a cold environment in which the temperature isin the range of 0.5° C. to 10° C. for a time period up to 6 months; (c)placing the germinants in water for a time greater than about 1 hour ata temperature below about 24° C.; and (d) removing the germinants fromthe water and transplanting them into growth medium for further growth.The methods of this aspect of the invention can be used to increase theconversion percentage from somatic embryos to plants growing in growthmedium for any conifer species, such as members of the family Pinacea,including members of the genus Pinus (e.g., loblolly pine (Pinustaeda)), or such as members of the genus Pseudotsuga (e.g., Douglas-fir(Pseudotsuga menziesii)).

In some embodiments, the germinants are stored on sterile germinationmedia for a time period from about 1 week up to 6 months, such as from 2weeks to 20 weeks, or more preferably from about 2 weeks to about 4weeks. In some embodiments, the germinants that have been pre-stored ongermination media and then stored in water for a time period from about1 hour up to 2 weeks, such as from about 1 week to about 2 weeks, morepreferably about 1 week.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

Example 1

This Example describes the results of an experiment demonstrating that atwo-step storage treatment on Douglas-fir germinants increases thesterile storage period without causing detrimental effects to greenhousesurvival.

Methods and Materials:

Germinants from ten different genotypes of Douglas-fir somatic embryoswere produced up to the development stage using the methods described inGupta, U.S. Pat. No. 5,563,061 and Welty, U.S. Pat. No. 6,134,830.Following the development stage, the cotyledonary embryos were placed onan agar gelled sterile germination medium, BM6, in Petri dishes for 4-12weeks under environmental conditions of 21° C. to 27° C. until goodepicotyl development was apparent (e.g., an epicotyl stem length ofabout 10 mm or greater, such as at least from 10 mm to 20 mm, such as atleast 12 mm, at least mm or greater). DM6 medium is a basal mediumlacking growth hormones which has been modified by reducing sucrose,myo-inositol and organic nitrogen.

Once good epicotyl development was apparent, the closed germinationcontainers containing the germinants on sterile germination medium weremoved directly into a cooler at a temperature of about 4° C. and storedin the dark for various amount of time up to 6 months. At various timepoints after storage on germination media, the germinants were removedfrom the germination media and placed into Petri dishes that werehalf-filled with Millipore or Nanopure water and then sealed withparafilm. The roots of the germinants were immersed in water while theshoots floated on the surface. The germinants in water were transferredto a cold environment at a temperature of about 4° C. for a period oftime up to 4 weeks.

Although the germinants were stored under sterile conditions up to thispoint, it was determined that the transfer, under a laminar flow hood,from the germination media to the Petri dishes containing water couldresult in desiccation of the germinants. Consequently, the transfer fromgermination media to water storage was not made under sterileconditions.

Germinants from ten different genotypes of Douglas-fir were treated asfollows. Group I control samples were germinated on sterile germinationmedia, then removed from the germination media and transplantedimmediately into potting media without a period of storage. Group IIgerminants were germinated on sterile germination media in germinationboxes, and the germination boxes were then moved into a dark cooler at4° C. for 1 week followed by one week in water storage. Group IIIgerminants were germinated on sterile germination media in germinationboxes, the boxes were moved into a dark cooler at 4° C. for 2 weeks,followed by one week in water storage. Upon removal from storage, thePetri dishes and boxes were inspected for signs of contamination. Nocontamination was found during the following experiments. The germinantswere transplanted into potting media and the percent survival wasmeasured at 8 weeks post transplant. The potting media consisted ofequal parts of peat, vermiculate and perlite. The percent survival ofthe germinants treated under the various conditions are shown below inTABLE 2.

TABLE 2 Douglas-fir Percent Survival Post-transplant Time held inDouglas-fir Genotype (percent survival 8 weeks post-transplant)Treatment Paired storage 7306 7435 3637 5022 5008 5010 5002 7667 6637744 Average Test Control (no 64.3 22.6 67.1 60.5 46.1 37.3 32.3 91.753.9 89.2 56.5 0.813 storage) 1 week on 63.5 13 51.4 67.2 41.1 40.5 48.1100.0 51.6 90.5 56.7 Germination media + 1 week in H₂O 2 weeks on 72.621.7 59.3 72.3 48.6 39.4 36.3 96.3 38.8 88.0 57.3 Germination media + 1week in H₂O

Results:

As shown above in TABLE 2, greenhouse survival post transplant forgerminants stored on germination media followed with one week of storagein water was at least as good, and in some cases better than thatobserved for water storage alone.

Conclusion:

The method of long term storage (e.g., 2 to 4 weeks, and likely up to 6months) of Douglas-fir germinants on germination media followed by shortterm storage (e.g., 1 to 2 weeks) in water results in improved survivalrates for transplants and provides a convenient method by which toaccumulate germinants prior to transplant. This method provides severaladvantages including increased survival post-transplant, convenientstorage of large numbers of germinants, and a reduced risk of microbecontamination prior to transplantation.

Example 2

This Example describes experiments that measure the post-transplantsurvival of various loblolly pine genotypes after storage of germinantsin water for various lengths of time as compared to the post-transplantsurvival of germinants that were stored on germination media followedwith one week of storage in water.

Methods and Materials:

Germinants Stored in Water

A control experiment was carried out to determine the effect of storagein water for various time periods on the post-transplant survival ofloblolly pine clones. Germinants from three different loblolly pinegenotypes were produced up to the germination stage using the methodsdescribed in Gupta, U.S. Pat. No. 5,563,061 and Welty, U.S. Pat. No.6,134,830. The germinants for each clone were removed from germinationmedia and placed into Petri dishes that were half filled with Milliporeor Nanopure water and then sealed with parafilm. The Petri dishes werestacked in a labeled crisper (1 genotype to crisper) and then put into adark walk-in cooler (approximately 4° C.) for the length of timeindicated below in TABLE 3 prior to transplant. A control batch ofgerminants from each clone was transplanted in potting mediumimmediately without storage in water. The potting medium consisted ofequal parts of peat, vermiculite, and perlite. The potted germinantswere held for further growth in a greenhouse maintained at 24° C. for a16 hour light period and at 18° C. for an 8 hour dark period. After 8weeks or more the plants were rated for survival percentage. The resultsare shown below in TABLE 3.

Germinants Stored on Germination Media Followed by Short Term Storage inWater

Germinants from different loblolly pine genotypes were produced up tothe germination stage using the methods described in Gupta, U.S. Pat.No. 5,563,061 and Welty, U.S. Pat. No. 6,134,830. The germinants fromthe different genotypes were stored on sterile germination media in thedark at 4° C. for various time periods, as shown in TABLE 4, then thegerminants were removed from germination media and placed into Petridishes that were half-filled with Millipore or Nanopure water and thensealed with parafilm and stored at 4° C. for a week prior totransplantation. The results ar-e shown in TABLE 4 and TABLE 5.

TABLE 3 Loblolly Pine Survival Post-Transplant After Storage in WaterLoblolly Pine Genotype Pr > t Time in (percent survival 8 or more Pairedtest Water weeks post transplant) 3 Clone 4 Clone (3 or 4 Storage 55 5668 75 Average Average genotypes) Control — 99.1 96 88.6 94.6 — 0.966(not stored) 1 week — 98.6 93.8 91.1 94.5 — Control 94.4 99.1 96 86.593.9 94.0 0.164 (not stored) 2 weeks 88.9 96.7 84.3 50.8 77.3 80.2Control — 98.8 96 87 93.9 — 0.860 (not stored) 3 weeks — 96.9 96 89.794.2 — Control 93.6 97.8 96.2 88.5 94.2 94.0 0.728 (not stored) 4 weeks95.7 96.5 98.0 83.3 92.6 93.4 Control — 98.3 96 86.6 93.6 — 0.147 (notstored) 5 weeks — 96.9 84 71.9 84.3 —

TABLE 4 Loblolly Pine Survival Post-transplant After Storage onGermination Media Followed by Storage in Water Loblolly Pine Genotype(percent survival 8 or more weeks post Time held in transplant) 3-ClonePr > t Storage 55 56 75 Average Paired test Control = 0 days 100 95.579.1 91.5 — on germination media + 21 days in H₂O 14 days on 100 93.477.6 90.3 0.195 germination media + 7 days in H₂O 21 days on 97.3 98.482.1 92.6 0.628 germination media + 7 days in H₂O Control = 0 days 100100 94.6 98.2 — on germination media + 21 days in H₂O 28 days on 10092.5 91.3 94.6 0.239 germination media + 7 days in H₂O

TABLE 5 Loblolly Pine Survival Post-transplant After Storage onGermination Media Followed by Storage in Water Time held TreatmentTreatment in Loblolly Pine Genotype Average Average Storage 202 205 212213 214 215 226 228 241 243 (10 genos) (4 genos) Control- 93.8 100 96.796.6 75 97.7 98 100 100 93.8 95.2 97.8 No Storage 2 weeks — — 100 96.3 —— 97.7 100 — — — 98.5¹ on Germ media + 1 week in H₂O 4 weeks 89.3 10095.9 98 89.8 100 93.6 98.1 93.3 96.3 95.4² 96.4³ on Germ media + 1 weekin H₂O ¹Paired t-test comparing 2-week Box Storage against the control(4 genos) (Pr > t) = 0.50 ²Paired t-test comparing 4-week Box Storageagainst the control (10 genos) (Pr > t) = 0.89 ³Paired t-test comparing4-week Box Storage against the control (4 genos) (Pr > t) = 0.32

Results:

As shown above in TABLE 2, the results indicate that short term waterstorage (e.g., 1 week) increases the post-transplant survival ofloblolly pine germinants, however longer term water storage (e.g., 2 to4 weeks or longer) decreases the percent survival in the majority ofclone genotypes (3/4) tested.

As shown above in TABLE 3 and TABLE 4, longer term storage ongermination media (e.g., from 2 to 4 weeks or longer) followed by shortterm (e.g., 1 week) water storage resulted in post-transplant survivalof loblolly pine germinants at survival rates at least as good as, andin some cases better than, germinants that were planted directly intopotting media with no storage.

Conclusion

The method of long term storage (e.g., 2 to 4 weeks up to 6 months) ofloblolly pine germinants on germination media followed by short termstorage (e.g., 1 to 2 weeks) in water results in improved survival ratesfor transplants and provides a convenient method by which to accumulategerminants prior to transplant.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method of producing and storing germinants prior to transplantationinto growth medium, comprising the steps of: (a) culturing conifersomatic embryos on sterile germination medium for a sufficient period oftime to produce germinants, said germinants comprising a visible,well-defined epicotyl and radical; (b) placing the germinants whilestill on sterile germination medium into a cold environment in which thetemperature is in the range of 0.5° C. to 10° C. for a time period up tosix months, and (c) placing the germinants in water at a temperaturebelow about 24° C. for a time greater than about one hour prior toplanting the stored germinants into growth medium.
 1. The method ofclaim 1, further comprising the step of planting the germinants intogrowth medium for further growth.
 2. The method of claim 1 in which thegerminants are Douglas-fir.
 3. The method of claim 1 in which thegerminants are loblolly pine.